RESEARCH NOTE

J. Food Sci. Technol. Nepal, Vol. 6 (110-113), 2010

ISSN: 1816-0727

Benzoic Acid Residue in Nepalese Fruits and Vegetable Products

KRISHNA PRASAD RAI*, SOVITA SHRESTHA, JIWAN PRAVA LAMA and BINAYA PRASAD SHRESTHA

Department of Food Technology and Quality Control, Kathmandu, Nepal

The research was carried out first time in Nepal to study the residue level of benzoic acid in fruits and vegetable products by
HPLC method. Altogether, 40 different samples of pickles, tomato ketchups, jam and some fresh condiments were analyzed. The
maximum concentration of benzoic acid (2192.4mg/kg) was found in pickles made in household level, while minimum quantity
(10.29mg/kg) was found in jam product. Altogether, 19.35% of samples analyzed were found to be contaminated with higher
quantity of benzoic acid as comparing to mandatory standard of Nepal. Individually, 33.33%, 10.00% and 11.11% of samples
were found to be substandard for pickles, tomato ketchups and jam respectively. Apparently, pickle samples were found to have
higher amount of benzoic acid as compare to others and no any evidence for natural contamination of benzoic acid in
vegetable products was observed. Furthermore, a simple water extraction method for sample preparation and new HPLC
procedure was established as a routine method for benzoic acid analysis.

Keywords: Benzoic acid, HPLC, Water extraction, Fruits and vegetable products

Introduction drink, fruit juice, margarine, yoghurt and cheese products

Benzoic acid (E210; benzene carboxylic acid; C7H6O2) and
its sodium (E211), potassium (E212) and calcium (E213) salts
(further referred to as ‘‘benzoates’’) are a group of food
additives important to preserve foods and to protect the
consumer from microbiological risks of various bacteria,
yeasts and fungi that can be involved in food poisoning,
such as Escherichia coli, Listeria monocytogenes,
Aspergillus spp. and Penicillium spp. (Sieber et al., 1995).
They are also used in other products except foods such as
pharmaceuticals and cosmetics. It has been estimated that
about 638,000 tons of benzoic acid has been produced
globally per year (WHO, 2000).
Due to its weak solubility in water (0.34g/100g), its sodium
salt i.e. sodium benzoate (63g/100g) is popularly used in fruits
and vegetable preservation (Kharel and Hasinaga, 2004). It
has a bacteriostatic and fungistatic effect under acidic
conditions pH below 4.0. Therefore, it is used most prevalently
in acidic foods such as salad dressings, carbonated drinks,
jams and fruit juices, pickles and other condiments. In Nepal,
the use of benzoic acid in different fruits and vegetable
products such as alcoholic and non alcoholic beverages, jam,
jelly, marmalade, pickles, preserve, fruit juice, tomato sauce,
ketchup and puree etc. has been limited by Food Regulation-
1971 (Rai, 2010). Therefore, the quantitative determination
thereof is warranted for routine analysis of these types of
product.
However a low quantity of benzoic acid has been detected in
some natural fruits and vegetables viz. cranberries, prunes,
greengage plums, cinnamon, ripe cloves and apples. A quite
significant level of benzoic acid has been detected in raw milk
in china (Qi et al., 2009). Similarly, different brands of soft
were found to be added a significant amount of benzoic acid
in Brazilian market (Tfouni et al., 2002).
JECFA (WHO, 1996) has evaluated and established an
acceptable daily intake (ADI) for benzoic acid, benzoate salt
(calcium, potassium and sodium), benzaldehyde, benzyl
acetate and benzyl alcohol of 0–5 mg/kg body weight. In
generally recognized as safe to a maximum level of use of
benzoic acid is 0.1% in the US and up to 0.25% in some other
developing countries. Because of varying diets among
countries, the foods that mainly contribute to the intake of
benzoic acid and its salt are expected to vary. Because of its
ability to cause contact urticaria it can provoke itching and
tingling in the mouth in sensitive subjects (Clemmensen and
Hjort, 1982). Some toxicological study claimed that benzoic
acid can damage and inactivate vital parts of DNA in a cell’s
mitochondria leading the disease like Parkinson’s and other
neurodegenerative diseases (Anonymous, 2010a).
Its toxicological data shows LD50 orally for mouse and rat as
1940mg/kg and 1700mg/kg respectively (RTC, 2010).
There are several quantitative determination techniques for
benzoic acid in food products viz. simple chloroform extraction
and gravimetric method, HPLC method, GC methods etc. (Lino
and Pena 2010; Qi et al., 2009; Tfouni and Toledo 2002; Ferreira
et al., 2000). Reviewing all the methods of analysis, this study
aimed to establish a new and cost effective water extraction
procedure for sample preparation and establish a specific
HPLC procedure for analyzing benzoic acid residue in fruits
and vegetable products available in Nepalese markets.

*Corresponding author, E-mail: krishnamalarai@yahoo.com

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 Rai et al., : J. Food Sci. & Technol. Nepal, 6 (110-113), 2010
Materials and Methods
Sampling: A purposive sampling technique was carried out
to collect randomly altogether 40 different fruits and vegetable
products available in Nepalese markets such as Hetauda,
Chitwan, Sunsari, Kathmandu, Kavere, Pokhara and Dhading
etc. All samples were packed in either glass jars or plastic
pouches and kept in refrigeration until the final analysis was
done.
Certified Reference Material (CRM) of benzoic acid was
purchased from RT Pharma, UK and an ISO Guide 34:2000
Reference material producer company.
Sample extraction and preparation: The samples were
extracted in water according to the following method
(Figure 1.)
Sample was mixed and grounded finely with the help of motor and pistol
Homogeneous sample was weighted approximately 10g
Samples was vertexed for 5min with 40ml of Milli-Q water
Samples are placed on water bath with reflux condenser at 700C for 45min
Samples are cooled down at RT and volume was made to 100ml with Milli-Q water
Samples are centrifuged at 6000rpm for 10min
The supernatant layer was filtered through Whatman No. 1 filter paper
Collected filtrate was diluted with Milli-Q water (As per requirement)
The final sample was placed in a Sonicator for 5 min
Sonicated samples were placed in HPLC vial (1.5ml) through syringe filter (0.22µm)
Any air bubbles and cloudiness were checked in sample if not vials were ready for injection.
The vials were placed in refrigeration till injected in HPLC
Figure 1. Water extraction method of sample preparation
for benzoic acid
Preparation of CRM standard: Firstly, 1000ppm of benzoic
acid stock solution was prepared in Milli-Q water (Milli-Q,
Academic, A10). A freshly 1, 2.5, 5 and 10 ppm of external
standards were prepared by dilution of stock sample with
Table 1. Recovery of benzoic acid from tomato ketchup and pickle
Sample Spiked amount (mg/kg)
Tomato ketchup 20
30
40
Pickle 20
30
40
Milli-Q water. The standards were vertexed well and kept for
5min in sonicator (PCI, India, 3.5L-100) before place in HPLC
vials through syringe micro filter (Milli-Q, 0.22μm). The
standard curve for concentration against peak area was plotted
and the coefficient of determinants (R2= 0.9991) was obtained
with equation y= 43816x, where the value ‘x’ is the
concentration of benzoic acid in sample.
Determination of Benzoic acid by HPLC: All the analysis
was carried out by using HPLC from Shimadzu equipped with
prominent degasser (DGU-20As), double pumps (LC-20AD),
Auto sampler (SIL-20A), detector, UV/VIS (SPD-20A), column
oven (CTO-20A) and LC solution software. The column used
was Pursuit C18 (Varian, 4.6x250. 10μm). The gradient mobile
phase consisted of 70:30 (0.02M phosphate buffer, pH 7.0:
Methanol) with the flow rate 0.8ml/min at 400C. The injection
volume was 10μl. The peak of benzoic acid was measured at
230nm. Chromatographic peaks were identified by comparing
retention time and spectra with the aqueous solution of the
standard. Quantification was based on the external standard
method and the concentrations of benzoic acid were 1.0, 2.5,
5.0, and 10.0 ppm. The best fit standard curve was prepared
by linear regression of peak areas versus concentration. The
determination of limit (LOD) was calculated, corresponding
to the analyte concentration equivalent to the blank signal
plus three standard deviations of the blank. The LOD for
benzoic acid was, respectively, 0.5 mg/kg in all products. The
analysis was conducted in duplicate.
Statistical analysis: The data obtained from analysis were
statistically analyzed by using the Microsoft Office Excel 2003.
Results and Discussion
Recovery: In order to verify the accuracy and precision of
analytical procedure, process recovery studies were carried
out. The known amount of benzoic acid (3 levels) was added
to the samples of ketchup and pickle. The mean recoveries
for ketchup and pickles were 100.40 % and 99.68%
respectively. Spiked samples were analyzed in triplicates.
Tfouni and Toledo (2002) have found a similar result of
recovery for pineapple fruit juice as 98.7% and for coca soft
drink as 102.2%.
Mean recovery ± SD
Recovery (%)
(%)
101.3 100.4±2.66
97.41
102.51
102.87 99.68±2.13
98.56
97.61
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 Rai et al., : J. Food Sci. & Technol. Nepal, 6 (110-113), 2010
Figure 4. Chromatogram of Chili powder
Conclusion
The study result revealed that 19.35% of fruits and vegetable
products were found to be contaminated with exceeding
quantity of Nepal government’s standard. Individually, 33.33,
10.00 and 11.11% of samples were found to be substandard
for pickles, ketchups and jam respectively. The maximum
residue level of benzoic acid (2,192.4mg/kg) was found in
pickles made in household level, while minimum quantity
(10.29mg/kg) was found in reputed brand for jam product.
Apparently, pickle samples were found to have higher amount
of benzoic acid as compare to others, which might be due to
the ignorance and lacking of facility for quality control
(unpublished survey data) in such household level industries.
Similarly, no any evidence for natural contamination of benzoic
acid in vegetable products was observed. Furthermore, a
simple and reliable water extraction method for sample
preparation and new HPLC procedure was established for
routine method for benzoic acid analysis.
Acknowledgements
This study was conducted according to the annual research
program entitled as ‘Study on benzoic acid residue in fruits
and vegetables products’ of DFTQC, Central Food Laboratory
in 2010. The first author is grateful to Mr. Ramesh Sthapit,
former DDG of DFTQC for giving me this opportunity and
also thankful to Ms. Helen Shrestha and Dipendra Ghimire
for sample collection and all the other staffs of CFL, who
directly and indirectly supports to complete this work.
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